1. Field of the Invention
The present invention relates generally to the field of cytology, and more particularly, it relates to an enhanced growth medium and method for isolating human mammary epithelial cells and growing such cells in both mass and clonal culture.
2. Description of the Prior Art
Studies on the cellular physiology of normal and pathologic human mammary epithelium have been hampered by the difficulty of growing these cells in vitro, free from other cellular elements of the mammary gland. While the short-term culture of epithelial cells derived from lactation fluids, reduction mammoplasties, benign tumors, non-tumor mastectomy tissue and primary carcinomas is known, in all cases the cells have displayed very limited potential for continued cell division and inability to maintain active cell growth upon serial subculture.
It would be particularly useful to be able to grow normal and malignant human mammary epithelial cells in both mass and clonal culture. For mass culture, it would be desirable to be able to increase the potential for cell division to be able to serially subculture cells from all specimens obtained, and to be able to cryopreserve cells from each individual donor for repetition of experiments. Such a cell culture system can be used for studies to develop an understanding of the physiology of normal and malignant human mammary epithelial cells and what agents might be capable of inducing normal cells to exhibit the properties of malignant cells.
A clonal culture where a small number of cells are plated and remain separate so that the percentage of individual cells that proliferate can be determined, may be used in a clonal assay to measure in vitro the cytotoxicity of particular noxious agents such as chemicals, chemotherapy drugs, radiation and the like. A clonal assay for human tumor cells was reported by Hamburger, et al., in an article entitled "Primary Bioassay of Human Tumor Stem Cells" SCIENCE 197: 461-463 (1977). In that procedure, individual cells suspended in agar enriched medium were plated into a petri dish having an agar feeder layer. While functional, this method suffers from several disadvantages. First, the plating efficiency is often lower than one colony per 10,000 cells plated. Second, there are difficulties in obtaining single cell suspensions, particularly with scirrhus tumors. Third, cryopreservation of primary tumor tissue has proven difficult. A clonal assay for measuring cytotoxicity of chemotherapeutic drugs is described in the paper "Quantitation of Differential Sensitivity Of Human-Tumor Stem Cells to Anticancer Drugs" by Salmon, et al., in the NEW ENGLAND JOURNAL OF MEDICINE, 298:1321-1327 ( 1978). The procedure described therein tested tumor cells from patients with myelomas and ovarian carcinomas for cytotoxic response for various chemotherapeutic drugs.
The most successful attempt at clonal growth of human mammary epithetal cells heretofore was reported by Sandback et al., in an article entitled "Assay for Clonogenetic Cells in a Human Breast Cancer" PROCEEDINGS AM. ASSOC. CANCER RES. 71:139 (1980) (using agar); and by Yang et al., "Effects of Hormones and Growth Factors on Human Mammary Epithelial cells on Collagen Gell Culture" CANCER RES. 41:1021-27 (1981) (using a collagen matrix). However, clonal growth was not obtained with either method unless more than 10.sup.5 cells were plated per dish.
There have been other reports of human mammary epithelial growth stimulated by feeder layers of fibroblastic (mouse and human) or bovine lens epithelial cells. See, Taylor-Papadimitriou, et al., "Growth Requirements of Human Mammary Epithelial Cells in Culture" INT. J. CANCER 20:903-908; and Kirkland, et al., "Growth of Normal and Malignant Human Mammary Epithelial Cells In Culture" JOURNAL OF THE NATIONAL CANCER INSTITUTE 63:29-42. Feeder layers formed from mouse or human mesenchymal cells are reported in Armstrong, et al., "Co-Cultivation Of Human Primary Breast Carcinomas In Embrionic Mesenchyme Resulting In Growth And Maintenance of Tumor Cells" CANCER RESEARCH 38:894-898. The use of such feeder layers in the absence of an adequate growth medium has not been able to extend the growth potential of pure cultures of human mammary epithelium to the extent desired.